The base excision repair pathway (BER) is responsible for detecting and repairing a wide array of oxidized and alkylated base lesions in DNA. These lesions are unavoidable as they arise from endogenous as well as exogenous sources; an individual cell may sustain as many as 10,000 lesions per day. BER is initiated by glycosylases, which locate and excise damaged bases. Thus, the specificity of the entire BER pathway is determined by the initiating glycosylase. Glycosylases face enormous challenges: they must be able to work without regard to sequence context; they must be able to recognize a wide variety of damaged bases; and they must effectively discriminate against undamaged bases. Using the human glycosylase Nth1 as a prototype, the proposed work will determine 1) the limits of the ability of Nth1 to work on all possible DNA sequences and 2) the mechanism by which Nth1 recognizes a wide array of oxidized pyrimidines, without erroneously excising undamaged bases. Accomplishing the first goal will identify sequences that are mutation hot spots because they are difficult to repair, and give insight into the evolution of the human genome. The second goal will identify the likely in vivo substrates of Nth1, allow estimation of the off-target activity of Nth1, and determine the probable outcome of Nth1 activity in cells. Finally, Nth1 has allelic variants in the human population, at least one of which is incredibly detrimental (Galick et al., 2013). The third goal of the proposed work is to determine the functional consequences of selected human variants. Accomplishing this goal will provide information about the mechanism of Nth1, as well as identifying defective Nth1 alleles that may have clinical utility.